Stem cell fate decisions to remain quiescent, self-renew or differentiate are largely governed by the interplay between extracellular signals from the niche and the cell intrinsic signal cascades and transcriptional programs. Here we demonstrate that DNA Damage Inducible Transcript 4 (DDIT4) acts as a link between HIF1&alpha; and mTOR signalling and regulation of adult stem cell fate. Global gene expression analysis of mesenchymal stem cells (MSC) derived from single clones and live RNA cell sorting showed a direct correlation between DDIT4 and differentiation potentials of MSC. Loss and gain of function analysis demonstrated that DDIT4 activity is directly linked to regulation of mTOR signalling, expression of pluripotency genes and differentiation. Further we demonstrated that DDIT4 exert these effects down-stream to HIF1&alpha;. Our findings provide an insight in regulation of adult stem cells homeostasis by two major pathways with opposing functions to coordinate between states of self-renewal and differentiation.

Mentions:
Excessive activation of mTOR signalling is suggested to drive unwanted differentiation and proliferation. mTOR activation has been shown to accelerate differentiation of MSCs and may result in depletion of the stem cell pool. Therefore we next examined the differentiation capacity of MSCs following manipulation of DDIT4 by either CoCl2 activation and gene knock-down or overexpression. Treatment with CoCl2 significantly reduced the expression of genes associated with osteoblast (Runx2 and ALP) and adipocyte (PPARγ) lineages in the absence of any differentiation stimulus (Fig. 3A), suggesting a reduction in spontaneous differentiation by MSC. Similarly when MSC were induced to differentiate in the presence of CoCl2, they showed a significant reduction in differentiation (Fig. 3B,C). Expression of markers of osteogenic (Fig. 3B) and adipogenic (Fig. 3C) differentiation were significantly reduced and the ability of the cells to undergo matrix mineralization (Fig. 3B) or the transformation to lipid containing adipocytes (Fig. 3C) was strongly impaired. We also stably knocked down DDIT4 which resulted in accelerated MSC differentiation toward both lineages, as determined by elevated expression of genes associated with osteoblasts (Fig. 3D) and adipocytes (Fig. 3E), increased matrix mineralization (Fig. 3D) and formation of lipid containing adipocytes (Fig. 3E) in response to stimulation of differentiation. However, no significant effect was observed under basal conditions (Fig. S2G). Interestingly, transient overexpression of DDIT4 in MSC had only a negligible effect on differentiation, which may be due to the temporary nature of expression (Fig. 3F).

Mentions:
Excessive activation of mTOR signalling is suggested to drive unwanted differentiation and proliferation. mTOR activation has been shown to accelerate differentiation of MSCs and may result in depletion of the stem cell pool. Therefore we next examined the differentiation capacity of MSCs following manipulation of DDIT4 by either CoCl2 activation and gene knock-down or overexpression. Treatment with CoCl2 significantly reduced the expression of genes associated with osteoblast (Runx2 and ALP) and adipocyte (PPARγ) lineages in the absence of any differentiation stimulus (Fig. 3A), suggesting a reduction in spontaneous differentiation by MSC. Similarly when MSC were induced to differentiate in the presence of CoCl2, they showed a significant reduction in differentiation (Fig. 3B,C). Expression of markers of osteogenic (Fig. 3B) and adipogenic (Fig. 3C) differentiation were significantly reduced and the ability of the cells to undergo matrix mineralization (Fig. 3B) or the transformation to lipid containing adipocytes (Fig. 3C) was strongly impaired. We also stably knocked down DDIT4 which resulted in accelerated MSC differentiation toward both lineages, as determined by elevated expression of genes associated with osteoblasts (Fig. 3D) and adipocytes (Fig. 3E), increased matrix mineralization (Fig. 3D) and formation of lipid containing adipocytes (Fig. 3E) in response to stimulation of differentiation. However, no significant effect was observed under basal conditions (Fig. S2G). Interestingly, transient overexpression of DDIT4 in MSC had only a negligible effect on differentiation, which may be due to the temporary nature of expression (Fig. 3F).

Stem cell fate decisions to remain quiescent, self-renew or differentiate are largely governed by the interplay between extracellular signals from the niche and the cell intrinsic signal cascades and transcriptional programs. Here we demonstrate that DNA Damage Inducible Transcript 4 (DDIT4) acts as a link between HIF1&alpha; and mTOR signalling and regulation of adult stem cell fate. Global gene expression analysis of mesenchymal stem cells (MSC) derived from single clones and live RNA cell sorting showed a direct correlation between DDIT4 and differentiation potentials of MSC. Loss and gain of function analysis demonstrated that DDIT4 activity is directly linked to regulation of mTOR signalling, expression of pluripotency genes and differentiation. Further we demonstrated that DDIT4 exert these effects down-stream to HIF1&alpha;. Our findings provide an insight in regulation of adult stem cells homeostasis by two major pathways with opposing functions to coordinate between states of self-renewal and differentiation.